Calpains are cysteine proteases implicated in a wide variety of cellular processes such as cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. We propose to test the hypothesis that calpain-1 regulates platelet signaling by modulating the activity of PTP1B under the following specific aims: (1) Regulation of PTP1B by calpain-1. We will determine the status of PTP1B cleavage in the wild type and calpain-1 null mouse platelets upon activation using domain-specific antibodies and immune complex phosphatase assays. We will measure the degradation of PTP1B and specific activity of degraded fragments and investigate the effect of PTP1B on c-Src activation in platelets. These studies will help to clarify the role of calpain-1 in the regulation of PTP1B and provide an explanation for the increased tyrosine phosphatase activity in calpain-I null mouse platelets. (2) Mechanism of calpain-1 dependent beta-3 integrin interactions with the platelet cytoskeleton. We plan to determine the identity of PTP1B substrates in platelets by gel electrophoresis and mass spectrometry. Using beta-3 integrin cytoplasmic domain constructs carrying conservative and radical replacement of tyrosines at 747 and 759, we will determine their phosphorylation stoichiometry, susceptibility to dephosphorylation by PTP1B, and quantify their interactions with Shc, myosin, and newly identified PTP1B substrates. These experiments, assisted by the BIAcore technology, will help to clarify the downstream effectors of PTP1B that are critical for outside-in signaling in platelets. (3) Role of calpain-1 signaling in platelet spreading. Using calpain-1 null mice with pure C57BL/6J genetic background, we will investigate the mechanism of enhanced platelet spreading by identifying the potential PTP1B-independent substrates of calpain-1 in mouse platelets. We will also test whether the calpain-1 null mice with the pure C57BL/6J genetic background display abnormalities in the platelet secretion and in vivo thrombosis status. Together, these studies will help to clarify the role of calpain-1 in integrating common and divergent aspects of platelet signaling, reveal novel aspects of PTP1B regulation, and provide a better understanding of the mechanism of platelet aggregation and clot retraction pathways. Ultimately, these studies may lead to the development of new therapeutic strategies against thrombosis. PUBLIC HEALTH RELEVANCE: Calpains are cysteine proteases implicated in a wide variety of cellular processes such as cell proliferation, apoptosis, cell motility, and hemostasis. The proposed studies will clarify the role of calpain-1 in platelet signaling and provide a better understanding of the mechanism of platelet aggregation and clot retraction pathways. Ultimately, these studies may lead to the development of new therapeutic strategies against thrombosis.